The yeast Candida albicans (C. albicans) is one of the most pervasive fungal pathogens in humans. It has the capacity to opportunistically infect a diverse spectrum of compromised hosts, and to invade many diverse tissues in the human body. It can in many instances evade antibiotic treatment and the immune system. Although Candida albicans is a member of the normal flora of the mucous membranes in the respiratory, gastrointestinal, and female genital tracts, in such locations, it may gain dominance and be associated with pathologic conditions. Sometimes it produces progressive systemic disease in debilitated or immunosuppressed patients, particularly if cell-mediated immunity is impaired. Sepsis may occur in patients with compromised cellular immunity, e.g., those undergoing cancer chemotherapy or those with lymphoma, AIDS, or other conditions. Candida may produce bloodstream invasion, thrombophlebitis, endocarditis, or infection of the eyes and virtually any organ or tissue when introduced intravenously, e.g., via tubing, needles, narcotics abuse, etc.
Candida albicans has been shown to be diploid with balanced lethals, and therefore probably does not go through a sexual phase or meiotic cycle. This yeast appears to be able to spontaneously and reversibly switch at high frequency between at least seven general phenotypes. Switching has been shown to occur not only in standard laboratory strains, but also in strains isolated from the mouths of healthy individuals.
Nystatin, ketoconazole, and amphotericin B are drugs which have been used to treat oral and systemic Candida infections. However, orally administered nystatin is limited to treatment within the gut and is not applicable to systemic treatment. Some systemic infections are susceptible to treatment with ketoconazole or amphotericin B, but these drugs may not be effective in such treatment unless combined with additional drugs. Amphotericin B has a relatively narrow therapeutic index and numerous undesireable side effects and toxicities occur even at therapeutic concentrations. While ketoconazole and other azole antifungals exhibit significantly lower toxicity, their mechanism of action, inactivation of cytochrome P.sub.450 prosthetic group in certain enzymes, some of which are found in humans, precludes use in patients that are simultaneously receiving other drugs that are metabolized by the body's cytochrome P.sub.450 enzymes. In addition, resistance to these compounds is emerging and may pose a serious problem in the future.
There is a need in the art for an effective treatment of opportunistic infections caused by Candida albicans. Therefore, one object of the invention is to provide screening assays for identifying potential inhibitors of Candida albicans growth. Another object of the invention is to provide screening assays and to identify potential inhibitors of Candida albicans growth that are based on inhibition of transcription in this organism.
Synthesis of mRNA in eukaryotes requires RNA polymerase II and accessory transcription factors, some of which are general and act at most, if not all, promoters, and others of which confer specificity and control. Five general factors, a, b, d, e, and g, have been purified to homogeneity from the yeast S. cerevisiae, and have been identified as counterparts of human or rat factors, TFIIE, TFIIH, TFIID, TFIIB and TFIIF, respectively. These factors assemble at a promoter in a complex with RNA polymerase II to initiate transcription. Binding studies have shown that the order of assembly of the initiation complex on promoter DNA begins with factor d (TFIID), is followed by factor e (TFIIB), and then by polymerase and the remaining factors. Factors b (TFIIH), e (TFIIB) and g (TFIIF), however, bind directly to polymerase II, and as many as four of the five factors may assemble with the polymerase in a holoenzyme before promoter binding. The functional significance of interactions revealed by binding studies is not clear in that only a few percent of initiation complexes may give rise to transcripts.
Many aspects of transcription by RNA polymerase II are conserved between yeast and higher eukaryotes. For example, there is extensive amino acid sequence similarity among the largest subunits of the yeast, Drosophila and mammalian polymerase. Other components of the transcription apparatus, such as TATA-binding and enhancer binding factors, are in some instances interchangeable between yeast and mammalian in vitro binding or transcription systems. There are, nonetheless, significant differences between the two systems. TATA elements are located from 40 to 120 or more base pairs upstream of the initiation site of an S. cerevisiase promoter, and where these elements occur, they are required for gene expression. The fact that C. albicans genes function in S. cerevisiase suggests that it also uses the 40 to 120 base pair spacing between the TATA element and initiation site. In contrast, mammalian (as well as S. pombe) TATA elements and transcription start sites are only 25 to 30 bp apart, and deletion of a TATA element does not always reduce the frequency of transcription initiation, although it may alter the initiation site. There are also varying degrees of homology between transcription factor sequences from yeast and mammalian sources. Human and S. cerevisiae TFIIB's have 50-60% amino acid sequence identity, and also are not species interchangeable in supporting cell growth. Some of the multisubunit factors, such as RNA polymerase II, TFIIF and TFIID, contain different numbers of subunits in humans and yeast. The molecular weights of corresponding polypeptides differ in humans and yeast with sequences being present in a given yeast factor that are not present in its human counterpart, and vice versa.
Operative substitution of the same transcription factor in transcription systems of different yeast strains is not predictable. This is true despite a high degree of amino acid sequence identity among some transcription factors from different yeast strains. For example, the ability of a given transcription factor to support efficient and accurate transcription in a heterologous yeast strain is not predictable. Li et al. (1994, Science 263:805) tested the interchangeability of S. cerevisiae and S. pombe transcription factors in vitro, and report that many S. cerevisiae components cannot substitute individually for S. pombe transcription factors a, e, or RNA polymerase II, but combinations of these components were effective. In one instance, active transcription could not be reconstituted when S. cerevisiae-derived TFIIB was the sole substitution into a TFIIB-depleted set of factors from S. pombe. A TFIIB-RNA polymerase II combination from S. cerevisiae was able to substitute, indicating that the functional interaction of these two components is not only important, but also that the activity may be dependent on species-specific determinants that cannot be complemented by either component derived from a different organism. The unpredictability in making substitutions of a given factor among different yeast strains is also evident in that such substitutions are not reciprocal; that is, substitutions of S. pombe fractions into an S. cerevisiae transcription system are less effective than the reverse substitutions (Li et al., supra).
The yeast Candida albicans differs from most yeast strains in that it does not use the same genetic code that most organisms, whether mammalian or yeast, utilize. Santos et al. (1995, Nucleic Acids Research, 23:1481) report that the codon CUG, which in the universal code is read as a leucine, is decoded as a serine in Candida. Therefore, any CUG codon that is decoded in Candida albicans as a serine, would be decoded as a leucine in the transformed S. cerevisiae. Any gene containing a CUG codon would therefore be translated as different amino acid sequences in Candida albicans and S. cerevisiae. Such mistranslation may produce an inactive protein, since the amino acids serine and leucine have markedly different chemical properties and serine is known to be an essential residue in the active site of some enzymes. Replacement of leucine by serine at CUG encoded residues is a serious problem in the use of many reporter systems (e.g., .beta.-galactosidase, Chloramphenicol acetyltransferase, Flux) in Candida albicans. Previous experiments have shown that translation by Candida of CUG as serine instead of leucine often resulted in the production of inactive reporter proteins.
Another object of the invention is to provide an assay for screening for selective inhibition of Candida albicans growth and/or viability.
Yet another object of the invention is to provide a molecular target for inhibition of Candida albicans transcription or transcription initiation.